In our previous study, gas-phase hydrazinolysis was used to analyze the glycoform of the O-linked oligosaccharide of human serum IgA1. All O-linked oligosaccharide chains are known to be present in the hinge portion. However, the number of O-linked oligosaccharide chains on IgA1 remained unclear. In order to determine the number of linked sugar chains, we applied matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) to the hinge glycopeptide prepared from human serum IgA1. MALDI-TOFMS did not show clear peaks, probably due to the microheterogeneity of the structure of each sugar chain. However, elimination of peripheral sialic acid and galactose residues by sequential treatment with neuraminidase and beta-galactosidase gave clear mass spectra with several sharp peaks. On the basis of these spectra, we conclude that IgA1 prepared from normal human serum carries different numbers of sugar chains. There are two major populations, one contains five GalNAc residues and the other four GalNAc residues. On the other hand, the hinge glycopeptide prepared from myeloma IgA1 was composed mainly of one population containing four GalNAc residues. Earlier, we reported incomplete glycosylation of IgA1 isolated from the serum of an IgA1 myeloma patient. In this experiment, the presence of four O-linked oligosaccharides per heavy chain of IgA1 from a myeloma patient was found. The reason why only four out of five sites on the hinge glycopeptide were fully glycosylated in the IgA1 from the IgA1 myeloma patient is not clear.

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For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) [Iwase, Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem. Biophys. Res. Commun. 151, 422-428], a method for assaying enzyme activity was established. Using purified pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated from PGM were pyridylaminated, and the reducing terminal sugars of oligosaccharides larger than Gal beta 1-3GalNAc were analysed by h.p.1.c. The crude enzyme of endo-GalNAc-ase-S was prepared as an 80% (w/v) ammonium sulphate precipitate from the concentrated culture medium. The enzyme was partially purified by gel chromatofocusing and subsequent DEAE-Toyopearl chromatography. Endo-enzyme activity eluted around pI 4.8 on a gel chromatofocusing column and eluted with 0.19-0.25 M-NaCl on a DEAE-Toyopearl column. In the enzyme fraction obtained, no exo-glycosidases or proteases could be detected. The molecular mass of the enzyme was estimated as 105 kDa by gel filtration, and the optimum pH was 5.5. Endo-GalNAc-ase-S hydrolysed the O-glycosidic linkage between GalNAc and Ser (Thr) in 3H-labelled and unlabelled asialofetuin, liberating both the disaccharide (Gal beta 1-3GalNAc) and the tetrasaccharide [Gal beta 1-3 (Gal beta 1-4GlcNAc beta 1-6)GalNAc]. When endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. (endo-GalNac-ase-A) was incubated with 3H-labelled and unlabelled asialofetuin, only the disaccharide (Gal beta 1-3GalNAc) was liberated.

The endo-a-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-I 1242 (endo-GalNAc-ase-S) hydrolyzed the 0-glycosidic linkage between GalNAc and Ser (Thr) in fetuin, liberating oligosaccharides. The 0-linked oligosaccharides liberated from the fetuin with endo-GalNAcase-S were pyridylaminated following fractionation on a Bio-Gel P-4 column. The structure of the pyridylaminated 0-linked oligosaccharides from fetuin has been determined by reverse-phase HPLC and 600-MHz 'H-NMR spectroscopy. The chemical shifts and the coupling constants of pyridylaminated (PA) NeuAca2-3GalpI -3GalNAc were refined by computer simulation of the spectrum. The structures of NeuAca2-3Ga1/31-3(NeuAca2-6)GalNAc-PA and NeuActr2-3GaljZ1-3(NeuAca2-3Gal~l-4GlcNAc~1-6)GalNAc-PA were determined by their structural reporter groups.Keywords: 0-linked sugar chain; 0-glycosidase; NMR; HPLC ; fetuin.The endo-a-N-acetylgalactosaminidase from a culture medium of Streptomyces sp. OH-I 1242 (endo-GalNAc-ase-S) was found to be capable of liberating not only Galpl-3GalNAc but other larger neutral oligosaccharides through the hydrolysis of the 0-glycosidic linkage between the GalNAc and Ser(Thr) residues [I, 21. The substrate specificity of endo-GalNAc-ase-S was different from that of endo-a-N-acetylgalactosaininidase (endoGalNAc-ase), i.e. 0-glycanase, from Diplococcus pneumoniae (endo-GalNAc-ase-D [3 -51) and endo-a-N-acetylgalactosaminidase from Alcaligenes sp. (endo-GalNAc-ase-A) [6], which are both commercially available, and can hydrolytically cleave the a-N-acetylgalactosaminyl linkages of only Galpl-3Gal-NAcal-Ser/Thr in glycopeptides and glycoproteins to liberate the disaccharide moiety. The endo-GalNAc-ase-S has been partially purified, and its specific activity, molecular mass and some characteristics have been reported [2], but its action on the sialoglycoprotein has not been examined at all. Fetuin, leukosialin, glycophorin A, submaxillary mucin, intestinal mucin etc. [7-131 are sialoglycoproteins, which have 0-linked sialyl oligosaccharide5. The structure of the oligosaccharide moiety of these sialoglycoproteins has been partially clarified, and changes in the sialyl oligosaccharide with differentiation, malignant transformation and immunodeficiency have been extensively studied [7,[14][15][16][17][18][19][20][21]. To investigate the oligosaccharide structure of the mucin-type glycoproteins, the O-glycoside linkage was chemically cleaved through weak alkalinity obtained in the p-elimination reactions, and the liberated oligoCorrespondence to K. Hotta, Department of Biochemistry, School of Medicine, Kitasato University, 1-15-1, Kitasato, Sagamiharashi, Kanagawa, Japan 228 F u r +81 427 78 9372. Abbreviutions. PGM, pig gastric mucus glycoprotein; Np, p-nitrophenyl ; PA, pyridylaminated; endo-GalNAc-ase, endo-a-N-acetylgalactosaminidase.Enzymes. Endo-u-N-acetylgalactosaminidase (EC 3.2.1.97) ; sialidase (EC 3.2.1.18).saccharide-alditols were investigated by 'H-NMR spectroscopy [22]. The enzymic cleavage of the 0-glycosidi...

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